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Published online before print April 9, 2004, 10.1110/ps.03474704
Protein Science (2004), 13:1322-1330. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Conformational analysis of HAMLET, the folding variant of human {alpha}-lactalbumin associated with apoptosis

Annarita Casbarra1, Leila Birolo1, Giuseppe Infusini1, Fabrizio Dal Piaz1, Malin Svensson3, Piero Pucci1, Catharina Svanborg3 and Gennaro Marino1,2

1 Dipartimento di Chimica Organica e Biochimica and
2 Facoltà di Scienze Biotecnologiche, Università di Napoli Federico II, Napoli, Italy
3 Department of Microbiology, Immunology and Glicobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden

(RECEIVED October 7, 2003; FINAL REVISION January 12, 2004; ACCEPTED January 15, 2004)



Abstract

A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human {alpha}-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-{alpha}-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the {beta}-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the {beta}-domain of {alpha}-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

Keywords: HAMLET; {alpha}-lactalbumin; conformational analysis; H/D exchange; limited proteolysis

Abbreviations: CD, circular dichroism • H/D, hydrogen/deuterium • {alpha}-LA, {alpha}-lactalbumin • MS, mass spectrometry • ESMS, electrospray mass spectrometry • MALDI-MS, Matrix Assisted Laser Desorption Ionization mass spectrometry • LCMS, liquid chromatography mass spectrometry • MS/MS, tandem mass spectrometry

Reprint requests to: Gennaro Marino, Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Room 2Mb-22, Via Cinthia, I-80126 Napoli, Italy; e-mail: gmarino{at}unina.it; fax: 39-081-674313.

Article published ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03474704.


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